The major objective of this R21 application is to demonstrate the feasibility of using an emerging biophotonics technology - partial-wave spectroscopic microscopy (PWS) for the diagnosis and surveillance of cholangiocarcinoma (CCA). CCA is a primary malignancy in bile duct epithelium. Although it is a rare cancer, the global incidence of this cancer is on the rise. It is a deadly disease with a median survival of months. However, significant improvements in survival rates are possible when the cancer is diagnosed at an early localized stage. Current imaging techniques suffer from either low sensitivity and sometimes lead to significant complications such as pancreatitis and cholangitis. The histopathological confirmation is notoriously difficult, with a suboptimal sensitivity of 9% up to 67% even when the advanced molecular techniques (e.g., fluorescence in situ hybridization (FISH)) were applied. These low rates are in large part due to the challenges of identifying malignant cells with conventional microscopic techniques in the setting of inflammation, and highlight a potential role for a novel biophotonic approach to diagnose malignancy by detecting subtle changes within a cell. PWS represents a novel molecular analysis of cell nano-architectural refractive index fluctuation arising from the changes in the concentration of intracellular solids (DNA, RNA and proteins, etc.) at a single cell level beyond what conventional microscopy reveals. We have completed successful pilot human studies to demonstrate the feasibility of PWS to improve the diagnostic accuracy and stratify risk for pancreatic cancer. This study will be guided by three specific aims: Aim 1 will assess the ability of PWS to improve the diagnostic accuracy of cytology by identifying CCA in patients with suspected biliary strictures in a retrospective study using archival cytologic specimens. Aim 2 will validate the optical signatures from PWS identified in Aim 1 in a prospective human study in an independent set of human subjects. Aim 3 will demonstrate the potential of PWS to diagnose as well as stratify risk for CCA through the identification of the "field-effect" of CCA, a concept of detecting a neoplastic alteration far away from the lesion, through the assessment of the duodenal epithelial cells in the vicinity of the bile duct. We believe that reasonable outcomes of the proposed project are to demonstrate the possibility of our optical technique to identify the presence of CCA in the duodenum and to improve the cytologic diagnosis of CCA. The long term goal is to develop a minimally invasive surveillance strategy to identify high risk individuals in whom intensive examination using more invasive procedures is warranted to detect a CCA at a stage when curable treatment is available, without the need for direct interrogation of the bile duct.